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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02768

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Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum

Vincenza Campana^1,2,*, Daniela Sarnataro^1,*, Carlo Fasano², Philippe Casanova², Simona Paladino¹ and Chiara Zurzolo^1,2,

¹ Dipartimento di Biologia e Patologia Cellulare e Molecolare and CEINGE, Centro di Biotecnologie Avanzate, Università degli Studi di Napoli `Federico II', via Pansini 5, 80131 Napoli, Italy
² Unité de Trafic Membranaire et Pathogénèse, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris CEDEX 15, France

View larger version (49K): [in a new window]   Fig. 1. PrPT182A acquires scrapie-like characteristics in transfected FRT cells. (A) PIPLC assay. FRT cells were grown on 60 mm dishes and lysed in Triton X-114 buffer. Aqueous phases were accurately recuperated and TCA precipitated, while detergent phases were incubated for 1 hour at 37°C in the presence (+) or absence (–) of PIPLC (5U/sample). 2% Triton X-114 was added and the samples were incubated for 10 minutes on ice. Separation was repeated and detergent (D) and aqueous (A) phases were recovered separately, immunoprecipitated and revealed by western blot. D, diglycosylated PrP; M, monoglycosylated PrP; U, unglycosylated PrP. (B) Triton/DOC insolubility assay. After lysis in Triton/DOC buffer, lysates of FRT cells were ultracentrifuged to separate detergent-soluble (S) and detergent-insoluble (P) molecules. The proteins were TCA precipitated and PrPs were separated by SDS-PAGE and analysed by western blot. (C) Proteinase K (PK) digestion assay. FRT cells were lysed in Triton/DOC buffer in the absence of protease inhibitors, and treated where indicated (+) with PK (3.3 µg/mg of protein) at 37°C for 2-10 minutes. The proteins were then TCA precipitated, separated by SDS-PAGE and immunoblotted. Note that the ratio between the amount of total proteins loaded in PK-untreated/PK-treated samples is 1:3.

View larger version (48K): [in a new window]   Fig. 2. PrPwt and T182A mutant acquire different oligosaccharide modifications in FRT cells. (A) FRT cells expressing PrPwt and PrPT182A mutant were lysed for 20 minutes in Triton/DOC buffer. PrPs were either untreated (control lanes) or digested for 16 hours with 5 mU/sample Endo-H (lanes H) or neuraminidase (lanes N). After TCA precipitation, proteins were revealed by SDS-PAGE and western blot with PRI308 antibody and chemiluminescence. D, diglycosylated PrP; M, monoglycosylated PrP; U, unglycosylated PrP. (B) FRT cells expressing PrPT182A were pulse-labelled with [35S]methionine for 20 minutes and then chased in medium containing unlabeled methionine for the indicated times. Cells were then lysed in Triton/DOC buffer and PrPT182A was immunoprecipitated. Half of the samples were treated with Endo-H (+) and half was left untreated (–) prior to analysis by SDS-PAGE and phosphorimager scanning.

View larger version (117K): [in a new window]   Fig. 3. PrPwt and T182A mutant have different intracellular distributions in FRT cells. (A) FRT cells expressing PrPwt and T182A were grown on transwell filters for 4 days and fixed with 2% paraformaldehyde. Saponin permeabilized (P) or non-permeabilized (NP) samples were treated with the -PrP antibody (PRI308) and the secondary TRITC-conjugated antibody. The samples were then examined in a Zeiss laser scanning confocal microscope (LSCM 510). Z (upper panels) and horizontal (lower panels) sections are shown. Bar, 10 µm. (B) FRT cells expressing PrPwt (left panels) and T182A (right panels) were grown on coverslips in semiconfluent conditions, fixed and permeabilized with 0.075% saponin. Then they were incubated with the -PrP mAb (PRI308) and with primary polyclonal antibodies against different markers of intracellular compartments, e.g. calnexin (CNX), giantin and early endosomal antigen 1 (EEA1) and then treated with -mouse and -rabbit secondary antibody conjugated with TRITC or FITC. Lysotracker was used to label lysosomes for 1 hour in vivo before fixation and confocal imaging. Bar,10 µm. Note that in the semiconfluent cells used for these experiments, which are optimal to visualize intracellular compartments, no plasma membrane signal was found for PrPwt (B). By contrast in polarized filter grown cultures the majority of the protein appears to be at the cell surface (A).

View larger version (54K): [in a new window]   Fig. 4. The untranslocated precursor of PrPT182A is degraded by the proteasome and ubiquitylated. (A) Steady-state proteasomal block. FRT cells expressing PrPwt and PrPT182A were treated with ALLN (150 µM) for 7 hours (+). The cells were then lysed and blotted with an -PrP antibody. Note that in the presence of ALLN a new PrP-specific band of 22 kDa is visible (arrow). (B) Pulse-chase analysis. FRT cells expressing PrPT182A were pulse-labelled with [35S]methionine for 20 minutes and then chased in medium containing unlabelled methionine for the indicated times in control conditions and after ALLN treatment, which was maintained for the entire length of the experiment. After lysis, PrPT182A was immunoprecipited and analysed by SDS-PAGE and phosphorimager scanning. The amount of the protein was quantified by NIH image software and expressed as a percentage of the amount of protein rescued after pulse (chase time 0) and plotted as a function of the chase time. The data points were fitted to an exponential curve using a non-linear regression analysis. Squares: control samples; circles: ALLN-treated samples. (C) PK protection assay. Microsomes were prepared as described in Materials and Methods and either left untreated or incubated with 250 µg/ml PK in the absence or presence of Triton X-100. The proteins were TCA precipitated and PrP molecules were detected by western blotting. The arrow indicates the ALLN-induced form of PrP that is selectively digested with PK in the absence of detergent. (D) Detection of PrP ubiquitylation. Cells were lysed in denaturing conditions (see Materials and Methods) and PrPT182A was immunoprecipitated with SAF32 antibody. After reduction in ß-mercaptoethanol, the sample was split in two, run on SDS-PAGE and revealed by western blotting either with the SAF32 antibody or a specific -polyubiquitin antibody to reveal polyubiquitylated PrP molecules. (E) Duplicates of each sample shown in D were run in parallel and then subjected to another run in a second dimension in SDS-PAGE. PrPT182A was revealed by western blotting either using -prion or -polyubiquitin antibodies. M, monoglycosylated PrP; Ut, untranslocated PrP; *, immunoglobulin chains and >>, polyubiquitylated PrP.

View larger version (68K): [in a new window]   Fig. 5. Proteasome block does not lead to cytosolic accumulation of PrPT182A nor to an increase in PK-resistant scrapie-like forms. (A) Immunofluorescence. FRT cells expressing PrPT182A were treated with ALLN for 7 hours, fixed and permeabilized with 0.075% saponin. Then, they were treated with the -PrP mAb (PRI308) and with primary polyclonal antibodies against CNX and giantin and with -mouse and -rabbit secondary antibodies conjugated with TRITC or FITC. The samples were then examined using a Zeiss laser scanning confocal microscope (LSM 510). Cytosol/membrane fractionation. (B) FRT cells expressing PrPT182A were treated or not with ALLN and cytosol (C)/membrane (M) separation was performed as described in Materials and Methods. Proteins were TCA precipitated and PrP molecules were revealed by western blotting. (C) After cytosol (C)/membrane (M) separation, PrPT182A was immunoprecipitated and revealed by SDS-PAGE and western blotting using either -PrP (SAF32) or -polyubiquitin antibodies (left and middle panels). As total control, 1/3 of total lysates was TCA precipitated and blotted by using the anti-polyubiquitin antibody (right panel). (D) Proteinase K (PK) digestion assay. Control and ALLN-treated cells were lysed and proteins were digested (+) or not (–) with PK, as described in Materials and Methods. TCA precipitation of PrP molecules were revealed by SDS-PAGE and western blotting. Note that the ratio between the amount of total proteins loaded in PK untreated/PK treated samples is 1:3.

View larger version (37K): [in a new window]   Fig. 6. Depletion of cholesterol perturbs PrPT182A raft association and increases its scrapie-like conversion. (A) Sucrose density gradients. FRT cells expressing PrPT182A were grown on 150 mm dishes in control conditions (control) or after cholesterol depletion (Mev/ß-CD). After lysis in 1% Triton X-100, 2 mg of total proteins were run through a two-step (5-30%) sucrose density gradient, as described in Materials and Methods. Twelve fractions were collected from the top to bottom of the tube after centrifugation to equilibrium, and PrPT182A was revealed in each fraction by western blotting. (B) Pulse-chase analysis in cholesterol-depleted cells. FRT cells expressing PrPT182A were cholesterol depleted (Mev/ß-CD) or untreated (control). After pulse-labelling with [35S]methionine for 20 minutes, they were chased in medium containing unlabelled methionine for the indicated times. Cells were then lysed and PrPT182A was immunoprecipitated and analysed by SDS-PAGE and phosphorimaging (left). The amount of protein was quantified by NIH image software and expressed as a percentage of the protein amount rescued after pulse (chase time 0) and plotted as a function of the chase time (right). The data points were fitted to an exponential curve using a nonlinear regression analysis. Squares, control samples; circles, Mev/ß-CD-treated samples. (C) PK digestion assay in cholesterol-depleted cells. PrPT182A-transfected FRT cells were grown in control (control) or cholesterol depleting (Mev/ß-CD) conditions. Cells were lysed in Triton/DOC buffer, in the absence of protease inhibitors, and were treated with PK (+) for 2 minutes. After SDS-PAGE, the PrPT182A mutant was revealed by western blotting. Data from different experiments were quantified using NIH image software for Macintosh as indicated in the results.